Hypoglycaemic peptides and methods of use thereof

ABSTRACT

A peptide of the formula (Xaa) n1 -Xaa 1 -His-Thr-Asp(Xaa) n2 , wherein Xaa is any amino acid; Xaa 1  is a hydrophobic amino acid, preferably Gly or Val; n1 is 0-10; and n2 is 0-10; and use thereof in regulating in vivo blood glucose levels in a human or other mammal, particularly in the treatment of Type 2 diabetes in a human. Preferably, the peptide is a tetrapeptide selected from Gly-His-Thr-Asp and Val-His-Thr-Asp. These hypoglycaemic peptides are isolated from human urine and they also have been chemically synthesized.

FIELD OF THE INVENTION

The present invention relates generally to a class of hypoglycaemicpeptides. More particularly, this invention relates to a method forregulating blood glucose levels of a human or other mammal byadministration of a peptide of this class. These hypoglycaemic peptidestherefore have potential for use as anti-diabetic agents, particularlyin treatment of Type 2 diabetes (or non-insulin-dependent diabetesmellitus, NIDDM).

BACKGROUND OF THE INVENTION.

Bibliographic details of the publications referred to hereinafter inthis specification are collected at the end of the description.

The reference to any prior art in this specification is not, and shouldnot be taken as, an acknowledgment or any form of suggestion that thatprior art forms part of the common general knowledge in Australia.

Type 2 diabetes results in chronic hyperglycaemia, hyperinsulinemia,insulin resistance, impaired insulin secretion and the risk ofcardiovascular complications (1-4). A recent report (5) showed thatthere are more than 150 million people worldwide who suffer fromdiabetes mellitus, among which over 90% of the people have Type 2diabetes. Currently, apart from insulin, no molecule of biologicalorigin has been found useful for the treatment and management of Type 2diabetes without further aggravating hyperinsulinemia which is believedto be a potential cause for the development of diabetes complications.

The isolation of a peptdic factor from human pituitary growth hormoneextracts which accelerated glucose uptake in isolated rathemi-diaphragms has been reported (6,7,8). The structure studiesdemonstrated that the molecule was a fragment of the amino terminalsequence of the growth hormone molecule. The amino-terminal region ofhuman growth hormone (hGH) containing the amino acid sequenceLeu-Ser-Arg-Leu-Phe-Asp-Asn-Ala (hGH 1-15) was found to enhance theactions of insulin in vitro and in vivo (9). This human growth hormonepeptide was used for used for comparison in the course of the work onthe urinary peptide factors.

Hypoglycaemic action of a semi-purified fraction of human urine has alsobeen observed (10,11). This urinary fraction acted only in the presenceof insulin in enhancing glucose uptake, glycogen synthesis, and glycogensynthetase conversion to the active form in vitro and in vivo. Thesimilar in vitro or in vivo biological effects of this urinary fractionled to the assumption that it was the hGH (6-13) fragment of humangrowth hormone, although no unequivocal evidence was obtained toestablish its identity. Studies with ultrafiltration, ion exchange andgel filtration chromatography indicated that the isolate from humanurine was a peptidic compound (11).

In work leading to the present invention, the hypoglycaemic peptide inhuman urine has been isolated and purified, and its structuredetermined. In addition, this peptide has been chemically synthesised.The activity of both the isolated peptide and the chemically synthesisedpeptide have been examined in vitro and in vivo to demonstrate itsinsulin-potentiating effects by enhanced glucose uptake and glycogensynthesis in vitro and lowered blood glucose levels in vivo. Inaddition, peptide analogues of this isolated peptide have also beenchemically synthesised, and certain of these analogues have also beenshown to have significant biological effects on glucose metabolism bothin vitro and in vivo.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a peptide of the formula:(Xaa)_(n1)-Xaa₁-His-Thr-Asp-(Xaa)_(n2)wherein

-   -   Xaa is any amino acid;    -   Xaa₁ is a hydrophobic amino acid;    -   n₁ is 0-10; and    -   n₂ is 0-10.

In a preferred embodiment of this aspect of the invention, the presentinvention provides a peptide of the formula:(Xaa)_(n1)-Gly-His-Thr-Asp-(Xaa)_(n2)or(Xaa)_(n1)-Val-His-Thr-Asp-(Xaa)_(n2)wherein Xaa, n₁ and n₂ are as defined above.

Preferably, the peptide is a tetrapeptide selected from:

-   -   Gly-His-Thr-Asp (hereinafter identified as UP-401); and    -   Val-His-Thr-Asp (hereinafter identified as UP-402).

In another aspect, the present invention provides the isolatedtetrapeptide Gly-His-Thr-Asp, in substantially purified form.

In a further aspect, the present invention provides a method ofregulating in vivo blood glucose levels in a human or other mammal,which comprises administration to said human or other mammal of aneffective amount of a peptide of the formula:(Xaa)_(n1)-Xaa₁-His-Thr-Asp-(Xaa)_(n2)wherein

-   -   Xaa is any amino acid;    -   Xaa₁ is a hydrophobic amino acid;    -   n₁ is 0-10; and    -   n₂ is 0-10.

In preferred embodiments of this aspect of the invention, the peptide isa peptide of the formula:(Xaa)_(n1)-Gly-His-Thr-Asp-(Xaa)_(n2)or(Xaa)_(n1)-Val-His-Thr-Asp-(Xaa)_(n2)wherein Xaa, n₁ and n₂ are as defined above.

Preferably, in this aspect of the invention, the peptide is atetrapeptide selected from:

-   -   Gly-His-Thr-Asp (UP-401); and    -   Val-His-Thr-Asp (UP-402).

In yet another aspect, the present invention provides use of a peptideof the formula:(Xaa)_(n1)-Xaa₁-His-Thr-Asp-(Xaa)_(n2)wherein

-   -   Xaa is any amino acid;    -   Xaa₁ is a hydrophobic amino acid;    -   n₁ is 0-10; and    -   n₂ is 0-10,        in the manufacture of a composition for regulating in vivo blood        glucose levels in a human or other mammal.

In preferred embodiments of this aspect of the invention, the peptide isa peptide of the formula:(Xaa)_(n1)-Gly-His-Thr-Asp-(Xaa)_(n2)or(Xaa)_(n1)-Val-His-Thr-Asp-(Xaa)_(n2)wherein Xaa, n₁ and n₂ are as defined above.

Preferably, in this aspect of the invention, the peptide is atetrapeptide selected from:

-   -   Gly-His-Thr-Asp (UP-401); and    -   Val-His-Thr-Asp (UP-402).

The present invention also provides a pharmaceutical composition forregulating in vivo blood glucose levels in a human or other mammal,which comprises the peptide of the formula:(Xaa)_(n1)-Xaa₁-His-Thr-Asp-(Xaa)_(n2)wherein

-   -   Xaa is any amino acid;    -   Xaa₁ is a hydrophobic amino acid;    -   n₁ is 0-10; and    -   n₂ is 0-10,        together with one or more pharmaceutically acceptable carriers        and/or diluents.

In preferred embodiments of this aspect of the invention, the peptide isa peptide of the formula:(Xaa)_(n1)-Gly-His-Thr-Asp-(Xaa)_(n2)or(Xaa)_(n1)-Val-His-Thr-Asp-(Xaa)_(n2)wherein Xaa, n₁ and n₂ are as defined above.

Preferably, in this aspect of the invention, the peptide is atetrapeptide selected from:

-   -   Gly-His-Thr-Asp (UP-401); and    -   Val-His-Thr-Asp (UP-402).

Preferably, in the peptides of this invention the amino acids are in theL-form, however the present invention also extends to peptides in whichone or more of the amino acids are in the D-, α- or β-form.

Throughout this specification, unless the context requires otherwise,the word “comprise”, and or variations such as “comprises” or“comprising”, will be understood to imply the inclusion of a statedinteger or step or group of integers or steps but not the exclusion ofany other integer or step or group of integers or steps.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a class of hypoglycaemic peptides whichmay be used to regulate in vivo blood glucose levels in a human or othermammal. Regulation of blood glucose levels, particularly lowering ofthese levels, provides a method for treatment of diabetes in humans,particularly treatment of Type 2 diabetes.

The peptides of the invention have the formula:(Xaa)_(n1)-Xaa₁-His-Thr-Asp-(Xaa)_(n2)wherein

-   -   Xaa is any amino acid;    -   Xaa₁ is a hydrophobic amino acid;    -   n₁ is 0-10; and    -   n₂ is 0-10.

Preferably, the hydrophobic amino acid is selected from the groupconsisting of Gly, Ala, Leu, lle, Phe or Val. Most preferably, thehydrophobic amino acid is Gly or Val.

Particularly preferred peptides of the invention are:

-   -   Gly-His-Thr-Asp (UP-401); and    -   Val-His-Thr-Asp (UP-402).        Preparation

The peptides of the invention as described above may be synthesisedusing conventional liquid or solid phase synthesis techniques. Forexample, reference may be made to solution synthesis or solid phasesynthesis as described in Chapter 9, entitled “Peptide Synthesis” byAtherton and Shephard, which is included in the publication entitled“Synthetic Vaccines” edited by Nicholson and published by BlackwellScientific Publications. Preferably, a solid phase peptide synthesistechnique using Fmoc chemistry is used, such as the Merrifield synthesismethod (12,13).

Alternatively, these peptides may be prepared as recombinant peptidesusing standard recombinant DNA techniques. Thus, a recombinantexpression vector containing a nucleic acid sequence encoding thepeptide and one or more regulatory sequences operatively linked to thenucleic acid sequence to be expressed may be introduced into andexpressed in a suitable prokaryotic or eukaryotic host cell, asdescribed, for example, in Gene Expression Technology: Methods inEnzymology, 185, Academic Press, San Diego, Calif. (1990), and Sambrooket al., Molecular Cloning: A Laboratory Manual, 2^(nd) ed. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

The UP-401 peptide may also be isolated from human urine by standardprotein purification procedures, preferably using reversed-phase highperformance liquid chromatography (RP-HPLC). Using these procedures,UP-401 is obtained in isolated form. By “isolated” is meant a peptidematerial that is substantially or essentially freed from components,particularly other proteins and peptides, that normally accompany it inits native state in human urine by at least one purification or otherprocessing step.

Such isolated UP-401 may also be described as substantially pure. Theterm “substantially pure” as used herein describes peptide material thathas been separated from components that naturally accompany it.Typically, peptide material is substantially pure when at least 70%,more preferably at least 80%, even more preferably at least 90%, andmost preferably at least 95% or even 99% of the total material (byvolume, by wet or dry weight, or by mole percent or mole fraction) isthe peptide of interest. Purity can be measured by any appropriatemethod, for example, in the case of peptide material, by chromatography,gel electrophoresis or HPLC analysis.

Formulations

The present invention also extends to pharmaceutical compositions forregulating in vivo blood glucose levels in humans or other mammals whichcomprise a peptide of the invention as described above, together withone or more pharmaceutically acceptable carriers and/or diluents.

The formulation of such therapeutic compositions is well known topersons skilled in this field. Suitable pharmaceutically acceptablecarriers and/or diluents include any and all conventional solvents,dispersion media, fillers, solid carriers, aqueous solutions, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like. The use of such media and agents forpharmaceutically active substances is well known in the art, and it isdescribed, by way of example, in Remington's Pharmaceutical Sciences,18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofaras any conventional media or agent is incompatible with the activeingredient, use thereof in the pharmaceutical compositions of thepresent invention is contemplated. Supplementary active ingredients canalso be incorporated into the compositions.

It is especially advantageous to formulate such compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the human or other mammalian subjects to be treated;each unit contains a predetermined quantity of active ingredientcalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier and/or diluent. The specificationsfor the novel dosage unit forms of the invention are dictated by anddirectly dependent on (a) the unique characteristics of the activeingredient and the particular therapeutic effect to be achieved, and (b)the limitations inherent in the art of compounding such an activeingredient for the particular treatment.

Administration

The present invention further extends to the methods for regulating invivo blood glucose levels in humans or other mammals by administering aneffective amount of a peptide of the invention as described above.Preferably, this treatment is administered to a human or other mammal inneed of therapeutic or prophylactic treatment for a disease condition orpotential disease condition. Most preferably, the treatment is treatmentof Type 2 diabetes in a human.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular condition beingtreated and the dosage required for therapeutic efficacy. The methods ofthis invention, generally speaking, may be practised using any mode ofadministration that is medically acceptable, meaning any mode thatproduces therapeutic levels of the active component of the inventionwithout causing clinically unacceptable adverse effects. Such modes ofadministration include parenteral (e.g. subcutaneous, intramuscular andintravenous), oral, rectal, topical, nasal and transdermal routes.

The active component may conveniently be presented in unit dosage formand suitable compositions for administration may be prepared by any ofthe methods well known in the art of pharmacy. Such methods include thestep of bringing the active component into association with a carrierand/or diluent which may include one or more accessory ingredients. Ingeneral, the compositions are prepared by uniformly and intimatelybringing the active component into association with a liquid carrier, afinely divided solid carrier, or both, and then, if necessary, shapingthe product.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the active component which ispreferably isotonic with the blood of the recipient. This aqueouspreparation may be formulated according to known methods using thosesuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in polyethylene glycol and lactic acid. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose, any bland fixed oil may be employedincluding synthetic mono-or di-glycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

Compositions of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets, tablets orlozenges, each containing a predetermined amount of the activecomponent, in liposomes or as a suspension in an aqueous liquor ornon-aqueous liquid such as a syrup, an elixir, or an emulsion.

Other delivery systems can include sustained release delivery systems.Preferred sustained release delivery systems are those which can providefor release of the active component of the invention in sustainedrelease pellets or capsules. Many types of sustained release deliverysystems are available; these include, but are not limited to: (a)erosional systems in which the active component is contained within amatrix, and (b) diffusional systems in which the active componentpermeates at a controlled rate through a polymer.

The active component is administered in therapeutically effectiveamounts. A therapeutically effective amount means that amount necessaryat least partly to attain the desired effect, or to delay the onset of,inhibit the progression of, or halt altogether, the onset or progressionof the particular condition being treated. Such amounts will depend, ofcourse, on the particular condition being treated, the severity of thecondition and individual patient parameters including age, physicalcondition, size, weight and concurrent treatment. These factors are wellknown to those of ordinary skill in the art and can be addressed with nomore than routine experimentation. It is preferred generally that amaximum dose be used, that is, the highest safe dose according to soundmedical judgement. It will be understood by those of ordinary skill inthe art, however, that a lower dose or tolerable dose may beadministered for medical reasons, psychological reasons or for virtuallyany other reasons.

Generally, daily oral doses of active component will be from about 0.01mg/kg per day to 1000 mg/kg per day. Small doses (0.01-1 mg) may beadministered initially, followed by increasing doses up to about 1000mg/kg per day. In the event that the response in a subject isinsufficient at such doses, even higher doses (or effective higher dosesby a different, more localised delivery route) may be employed to theextent patient tolerance permits. Multiple doses per day arecontemplated to achieve appropriate systemic levels of compounds.

Further features of the present invention are more fully described inthe following Example(s). It is to be understood, however, that thisdetailed description is included solely for the purposes of exemplifyingthe present invention, and should not be understood in any way as arestriction on the broad description of the invention as set out above.

IN THE ACCOMPANYING DRAWINGS:

FIG. 1 shows separation of a semi-purified urinary peptide on C18(A) andC3 (B, C and D) columns by four-step reversed-phase HPLC procedures.

FIG. 2 shows the mass spectrum of the urinary peptide UP-401.

FIG. 3 shows stimulation of glycogen synthase activity in isolated rathemi-diaphragm muscle tissue by insulin and by synthetic peptides UP-401and hGH (1-15) (0.1 μM).

FIG. 4 shows the in vivo effect of UP-401 peptide on glycogenmetabolism. Data represent the mean +/− SEM of five independentexperiments. The changes in blood glucose levels were statisticallysignificant (p<0.005) as compared with control.

FIG. 5 shows the HPLC profiles of peptides 1, 2 and 3 (see Table 2). Amixture of synthetic peptides (Gly-His-Thr-Asp, Val-His-Thr-Asp andAsp-His-Thr-Gly) and their corresponding β-isomerised formation(Gly-His-Thr-βAsp, Val-His-Thr-βAsp and βAsp-His-Thr-Gly) were separatedby reverse-phase HPLC and eluted with an isocritic running with 2%buffer B (buffer A is 0.1% trifluoroacetic acid in H₂O) at 1 ml/min over30 min. Peptides were determined by their absorbance at 214 nm.

FIG. 6 shows β-isomerisation of the N-terminal Asp-X sequence. Attack bythe peptide backbone nitrogen of the carbonyl group can result in theformation of a five membered succinimide ring. The succinimide ring isprone to hydrolysis, producing ring nonisomerised (Asp) and β-isomerised(βAsp) peptides in a ratio of ca. 1:3 when the site is Asp-His.

FIG. 7 shows stimulation of glycogen synthase activity in tissue byinsulin and by synthetic peptides UP-401, UP-401¹, UP-402, UP-402¹,UP-403, UP-403¹, UP-404 and hGH1-15.

FIG. 8 shows the in vivo effect of UP-401 peptide and analogues UP-402and UP-403 on glycogen metabolism. Data represent the mean +/− SEM offive independent experiments. The changes in blood glucose levels werestatistically significant (p<0.005) as compared with control.

EXAMPLE 1

In this Example, we have isolated and characterised the UP-401 peptidefrom human urine, chemically synthesised UP-401 and examined itsactivity in vitro and in vivo. Results suggest that UP-401 exhibitsinsulin-potentiating effects, as demonstrated by the enhanced glucoseuptake and glycogen synthesis in vitro and lowered blood glucose levelin vivo.

Materials and Methods

Materials

All chemicals and reagents used for peptide synthesis were purchasedfrom Auspep Pty.Ltd. (Melboume, Australia), except for ninhydrin testkit from PerElmer. Acetonitrile was purchased from MallinckrodtSpecialty Chemicals (Melbourne, Australia). 1,3-Diisopropylcarbodiimidewas from Aldrich Chemical Company (Milliford, Mass. U.S.A.). Insulin(100 U/ml) and bovine serum albumin (BSA) were obtained from theCommonwealth Serum Laboratories (Melbourne, Australia), D-glucose (A.R.)from BDH Chemicals Australia Pty. Ltd. (Kilsyth) and D-¹⁴C(U)glucosefrom NEN (Boston, USA).

Purification of Human Urinary Material

Urine specimens were collected from normal subjects and processed asdescribed by Ng, et al, (11). In brief, urine was acidified with HCl topH 2.1 and concentrated using the Diaflo ultrafiltration technique. Thecrude samples were chromatographed first on Dowex 50W-X2 and then onAmberlite CG50 columns. The fractions containing the biologically activematerial were eluted from the CG50 column with H₂O and lyophilised.

The crude urinary peptide material was further purified in a number ofconsecutive reverse-phase HPLC steps as described below.

The lyophilised semi-purified urinary material was dissolved in 0.1%trifluoroacetic acid (TFA) (v/v) and eluted with isocratic elution of0.1% TFA for 5 min and then a linear gradient of 50% acetonitrile usinga Vydec C18 column (4.6×250 mm). Seven fractions were collected,freeze-dried, redissolved in saline (0.9% NaCl) and assayed forbiological activity in vitro, using ¹⁴C-glucose incorporation into¹⁴C-glycogen in rat hemi-diaphragm muscle as an index.

Fraction 1 containing most of the biological activity was dissolved in0.1% trifluoroacetic acid and further purified using a Vydac C3 column(4.6×250 mm). The sample was eluted with an isocratic condition with amixture of solvents at a flow rate of 0.1 ml/min. The elutant was madeup with solvent A (0.1% TFA in H₂O) and solvent B (0.1% TFA in 50%acetonitrile). Three fractions were collected, freeze-dried, redissolvedin saline (0.9% NaCl) and assayed in vitro for biological activity asbefore. The first fraction again contained the most active material andwas subjected to re-purification on HPLC using a Vydac C3 column aspreviously described. This repeat HPLC run yielded the active purematerial in Fraction 3 which was then lyophilised for structure studies.

HPLC Equipment

RP-HPLC was performed on a Waters 600 E multisolvent delivery system(Eschbom, Germany) with a scanning Model 484 Tunable Absorbance detector(Millipore, USA) and an extended wavelength module equipped with a 214nm cut-off filter. The peptides were purified on a 3 μm Vydac C18(4.6×250 mm) and C3 (4.6×250 mm) columns. The effluents were monitoredfor peptide bonds by UV absorption at 214 nm at room temperature.

Peptide Sequence Analysis

N-terminal sequencing of the selected peptide fraction was performedusing an ASI 477 ‘pulse liquid’ protein sequencer with an on-line ABI120 A analyser (Applied Biosystems, Inc. Model 475 A).

Mass Spectrometry Analysis

The urinary peptide and the synthetic analogues at a concentration ofabout 1.0 mM were dissolved in an acetonitrile/water mixture (v/v). A 10μl sample was injected with a loop injector into a carried stream ofacetonitrile/water (50/50) pumped at 10 μl/min into the electrosprayion-source. Mass spectra were performed on a Fisons Instrument, Model VGPlatform II (Micromass Ltd., Cheshire, UK) micromass platforminstrument. The single charged pseudo-molecular ions (M+H)⁺ wereselected for collision-induced dissociation in the second quadruple andfor mass analysis of the daughter ions formed. The mass spectrometryexperiments were performed with electrospray with positive ion polarityand the cone voltage=70V @ 400 m/z to 30V @1200 m/z.

Peptide Synthesis

Peptide syntheses were performed by standard solid-phase peptidesynthesis method using Fmoc as the N-amino protecting group (12,14).Couplings were carried out using the DIC+HOBt method (15).

Animals

Male Wistar rats of 380-450 g (10 week) and male Zucker (fa/fa) fattyrats of 400-500 g (10 week) were used for the studies. The animals werehoused in the Departmental animal house under the conditions of constanttemperature with a 12h light, 12h dark cycle and fed ad libitum. Theanimals used in this investigation were cared for by trained animaltechnicians in the Monash University Department of Biochemistry andMolecular Biology. All experiments were approved by the StandingCommittee on Ethics in Animal Experimentation of Monash University.

In Vivo Biological Assay

In vivo tests of the hypoglycaemic peptides were performed onovernight-fasted male Zucker fatty (fa/fa) rats 45 min after inductionof anaesthesia with an intraperitoneal injection of sodiumpentobarbitone at a dose 60 mg/kg body weight. Basal blood glucosesamples were collected from the cut tip of the tail. The two femoralveins in the anaesthetised animals were then exposed, followedimmediately by an intravenous injection of saline (control) or peptideinto the left femoral vein. Approximately 15 min after theadministration of peptide or saline via the left femoral vein, insulinwas injected via the right femoral vein. Blood samples were taken at 5,10, 15, 30, 45, 60 and 90 min after injection of insulin. Blood glucoselevels of the collected samples were measured immediately by the glucoseoxidation method using a Yellow Springs YSI modal 2300 STAT GlucoseAnalyzer (Yellow Spring, Ohio).

In Vitro Biological Assay

Glycogen synthesis in isolated rat hemidiaphragm muscle was measured bythe rate of incorporation of radioactive glucose into glycogen (16).Overnight-fasted male albino Wistar rats (20 week old) were killed bydecapitation and their hemidiaphragm tissues immediately removed. Thehemidiaphragm tissues were pre-incubated for 30 min at 370° C., inKreb's-Ringer bicarbonate buffer (KRB) containing 1.0% bovine serumalbumin BSA, and 1.0 mg/ml glucose gassed pH 7.4. After pre-incubation,the tissues were further incubated for 60 min at 370° C. in 2 ml ofincubation buffer in the presence of insulin at a concentration 0.33μu/ml and the label ¹⁴C-glucose at 0.28 μCi/ml under an atmosphere ofcarbogen with or without peptide of various concentrations. At the endof the incubation, the tissues were removed and rinsed with colddistilled water, blotted and placed in a plastic centrifuge tubes forglycogen extraction with 30% KOH 10 μl/mg tissue for 5 min in a boilingwater bath. The tissue ¹⁴C-glycogen was precipitated with saturatedNa₂SO₄ and absolute alcohol. After centrifugation at 4000×G at 4° C. for10 min, the supematant was discarded and the precipitate washed twice byresuspending in 66% ethanol and centrifuged as before. The finalglycogen precipitate was dissolved in distilled water 30 μl/mg and 1.0ml of the solution was transferred to a counting vial containing atriton scintillator (8 ml). Radioactivity was determined in a Wallac LKBcounter from Pharmacia-Wallac Oy, (Turku, Finland). The total glycogencontent in the diaphragm muscle was calculated according to the methodof Van Handel (17).

Results

The highly purified peptide was obtained from human urine specimens withmultiple runs in reverse-phase HPLC. The bioassay of separated fractionswas determined at each step. In the first HPLC run, trifluoroacetic acidwas used as an ion-pairing agent. The urine specimens consisted of avariety of different hydrophilic and hydrophobic molecules as shown bythe elution profiles in FIG. 1A. Seven fractions were collected andassayed in vitro using glycogen synthesis in rat hemidiaphragm muscle asan index of bioactivity. Fraction 1 exhibited higher biological activityon glycogen synthesis than that of all other fractions and was selectedfor further purification. FIG. 1B shows the reverse-phase elutionprofile of the fraction in which five fractions were collected forassays in vitro.

The third fraction with the highest bioactivity was lyophilised andsubjected to further HPLC purification. The reverse-phase elutionprofile of the third fraction from B revealed several different peaks(Fig. 1C). Four fractions were also collected and assayed in vitro inwhich fraction 3, showing higher activity on glycogen synthesis thanthat of all other fractions, was lyophilised and subjected to a finalpurification for structure analysis. The purity of the urinary peptidewas assessed with amino acid sequence analysis and mass spectrometry.

Amino Acid Sequence Analysis of Purified Urinary Peptide

The N-terminal amino acid sequence analysis of the final pure urinarypeptide revealed that the primary structure is Gly-His-Thr-Asp. Thispeptide is hereinafter referred to as the UP-401 peptide.

Determination of Mass Spectrometry

The purity of the UP-401 peptide obtained by HPLC was assessed with massspectrometry. The UP-401 peptide in the positive ion mode revealed asingle predominant ion corresponding to the protonated molecule (M+H⁺)at m/z 429.8 with few impurities (FIG. 2). This molecular mass was 428Da from the final purification with HPLC (FIG. 1D) and exclusivelyresponsible for the size of sequenced fragment from amino acid sequenceanalysis. Reduction of protonated molecule (M+H⁺) to dehydrated moleculeresulted in a net decrease of 18 Da due to the loss of a hydrogenmolecule. Reduction of molecule weight 410 Da (Asp-His-Thr-Gly) to 353Da (His-Thr-Gly) resulted in a net decrease of 57 Da due to the loss ofan Asp residue. The molecule weight with m/z 429.8 Da was reduced to m/z274 Da due to the loss of a histidine as well as an Asp residue.

Based on the revealed structure of the UP-401 peptide, a syntheticpeptide corresponding to its sequence was made. The synthetic UP-401peptide was purified by RP-HPLC using a C18 column and confirmed by massspectrometry.

Biological Activity of Urinary Peptide Analogues

The biological action of the UP-401 peptide was examined and results areshown on FIG. 3. A 36.3% increase of glycogen synthesis by UP-401 wasdetermined, which showed a better insulin-potentiating effect than thatof the known insulin-potentiating hGH(6-13) peptide. Furthermore, the invivo effect on blood glucose level was observed after administration ofthe UP-401 peptide at a dose of 3 mg/kg body weight (FIG. 4). The invivo hypoglycaemic action of UP-401 in the male Zucker fatty (fa/fa)rats was found 15 minutes after the injection, with maximal effect 45minutes after treatment, and diminished after 90 minutes. Thehypoglycaemic effect of UP-401 was found significant when compared tothe control animals treated with saline.

Discussion

Although a crude fraction from human urine has been shown (10,11) tohave similar biological activity in vitro and in vivo to the syntheticpeptides containing the human growth hormone sequence 1-15 (hGH 1-15),no evidence was provided to indicate the molecular structure of theactive component. In this Example, techniques, such as HPLC, amino acidsequencing, and mass spectroscopy, have been used for the purificationand characterization of the structure of active compound, UP-401. Alldata suggest that the UP-401 is a tetrapeptide with the primarystructure Gly-His-Thr-Asp. The scientific evidence was further confirmedby making synthetic UP-401 and testing its biological activities.Results from the in vitro metabolic action on glycogen synthesis as wellas the in vivo animal trials on lowering blood glucose both demonstratedthe insulin potentiating-effect of the UP-401 peptide.

EXAMPLE 2

This study focuses on improving the biological potency of the UP-401peptide by studying the structure-actvity relationship. Peptideanalogues were designed and made by either replacing or isomerising theresidues of UP-401 and biological assays were conducted to test thepotency of each designed analogue.

Materials and Methods

Materials

See Example I.

Peptide Synthesis

Syntheses of peptide analogues were performed by a standard solid-phasepeptide synthesis method using Fmoc Chemistry (12). As in Example 1,couplings for peptide bond formation were carried out using the DIC+HOBtmethod (15) and purification was also conducted by reverse-phase HPLCmethodology.

Mass Spectrometry Analysis

The molecular structure of each peptide analogue was confirmed bydetermining its molecular weight, using the electrospray massspectrophotometer as described in Example 1.

In vitro and in vivo Biological Assays

Experimental procedures for the in vitro and in vivo assays weredescribed in Example 1. In principle, the rate of conversion fromglucose into glycogen in isolated hemidiaphragm muscle was used as theindex to determine the in vitro activity of peptides (6). The in vivotests for the peptides were performed on Zucker fatty (fa/fa) rats byi.v. administration of peptides and the reduction of the blood glucoselevels was measured and regarded as the index of the hypoglycaemiceffect (18).

Results

Based on the structure of the UP-401 peptide, analogues with varioussequences were synthesised for further biological characterization asshown in Table 1. All synthetic peptides were purified by RP-HPLC usinga C18 column and characterized by mass spectrometry. During synthesis,the peptide chains were treated with base (piperidine) in deprotectionsteps and then eventually treated with TFA at cleavage steps. TABLE 1The primary sequence of urinary peptide and the synthetic analoguesPeptide Analogues Sequence UP-401 Gly-His-Thr-Asp-NH₂ UP-402Val-His-Thr-Asp-NH₂ UP-403 Asp-His-Thr-Gly-NH₂ UP-404Val-His-Thr-Pro-NH₂ UP-405 Gly-His-Thr-isoAsp-NH₂ UP-406Val-His-Thr-isoAsp-NH₂ UP-407 isoAsp-His-Thr-Gly-NH₂

Table 2 and FIG. 5 outline the amino acid sequence of the UP-401 peptideand its analogues, and the ratio of nonisomerized and β-isomerisedpeptide obtained in each. TABLE 2 The primary sequence of urinarypeptide and the structures of urinary peptide analogues. Peptide No.Sequence Isoasp 1 Gly-His-Thr-Asp-NH₂ 45.2 2 Val-His-Thr-Asp-NH₂ 48.3 3Asp-His-Thr-Gly-NH₂ 75.2 4 Val-His-Thr-Pro-NH₂ —

It is clear from FIG. 5 that the ratio of β-isomerised and nonisomerizedpeptides (peptides 1 and 2) are closer, whereas in the case of peptide 3the ratio of β-isomerised and nonisomerized peptides increased to 3:1(FIG. 6). Then, it can be seen that peptide structure affects therelative ratios of isoaspartate formation from different sequences. Itseems important to evaluate whether sequence difference around thedifferent sites might provide an explanation for the different ratios ofisoaspartate formation (19,20,21,22).

Biological Activity of Urinary Peptide Analogues

The action of the peptide analogues in the regulation of glycogenmetabolism was examined. The experimental results (FIG. 7) on theinsulin-stimulated glycogen synthesis in isolated rat muscle indicatedthat the biological activity of Val-His-Thr-Asp was much higher thanthat of any other peptide, and the nonisoaspartate peptides were moreactive than the isoaspartate peptides. When the residue Asp of theVal-His-Thr-Asp peptide was isomerised to Val-His-Thr-βAsp, thebiological activity of this isoaspartate peptide, at a peptideconcentration of 0.1 mM, was reduced to 46%. The effects ofAsp-His-Thr-Gly and βAsp-His-Thr-Gly on glycogen synthesis were alsodetected. However, no appreciable stimulation was detected with thepeptide analogues Val-His-Thr-Pro in comparison with the control.

The in vivo effect on blood glucose level was observed afteradministration of the peptide (UP-401) at a dose of 3 mg/kg body weight.The maximal decrease in blood glucose level was found in the male Zuckerfatty (fa/fa) rats 60 min after treatment with UP-401 (3 mg/kg). Thedegree of blood glucose reduction varied with different peptideanalogues (Table 3). When the Val-His-Thr-Asp-NH₂ (UP-402) peptide wasadministered at a dose of 3 mg/kg body weight, the blood glucose levelwas reduced to a maximum of 0.95 mol/L. Increasing the dose ofVal-His-Thr-Asp peptide to 4.5 mg/kg body weight, the effect wasprolonged to 120 min (FIG. 8). Under the same conditions theVal-His-Thr-βAsp peptide was also tested for-its effect on blood glucoselevels. No significant difference between Val-His-Thr-Asp andVal-His-Thr-βAsp on the blood glucose levels was observed. TABLE 3Reduction of blood glucose level (in vivo insulin-like effects oftetrapeptide on glucose metabolism). Time 402 402 401 404 Control (min)(3 mg) (4.5 mg) (3 mg) (3 mg) (Saline) 15 −0.26 −0.43 −0.50 −0.21 −0.130 −0.62 −1.03 −0.91 −0.54 0.2 45f −1.21 −1.50 −1.1 −0.77 0.1 60 −1.25−1.45 −0.93 −0.95 0.1 75 −0.64 −1.52 −0.64 0.23 0 90 0.05 −1.35 −0.08−0.03 0.1 120 0.07Discussion

In this study, a series of synthetic peptide analogue of the UP-401peptide, from UP-402 to UP-407, were designed, prepared and tested toinvestigate the structure-activity relationship (SAR)

Results suggested that a hydrophobic amino acid at the position 1 couldsignificantly enhance the insulin-potentiating effect of the UP-401peptide. In vitro data indicated that a 68% higher glycogen synthesiswas found by the effect of UP-402, an analogue with the substitution ofGly by Val. On the other hand, replacement of Gly with the hydrophilicamino acid, Asp (UP-403) or isoAsp (UP-407), reduced the peptideactivity.

Results also suggested that the position 4 of the UP-401 peptide wascritical to biological activity of the UP-401. Analogues withreplacement of on Asp by another amino acid like Gly (UP-403), or Pro(UP-404) or even isoAsp (UP-406) all reduced the potency of the parentUP-401. The biopotency of UP-402, a better analogue, was also reduced bythe replacement of Asp by isoAsp, which further indicated that the Aspat the position 4 is essential for the biological activity. Thus, allthe evidence strongly suggested that the Asp⁴ residue plays an importantrole for the activity of UP-401, since changes by substitution andisomerisation reduced the potency of the molecule.

These investigations have also provided a better insight in the designand synthesis of structurally related Thr-Asp site active centresequences. It has been found that the ratio of nonisoaspartate withisoaspartate in the sequence of Gly-His-Thr-Asp was very similar to thatof sequence Val-His-Thr-Asp. Under the same condition a ratio ofnonisoaspartate and isoaspartate is 1:3 (FIG. 6), it might be that thenitrogen atom of the side of histidine residue could help deprotonationof the nitrogen in peptide bond, by achieving the necessary nucleophiliccharacter to form a succinimide product (22,23,24,25). On the otherhand, the degree of steric hindrance by the side-chain of the N+1residue is the crucial factor directly to effect the stability of themolecule (26) The nonisoaspartate peptide with Thr-Asp site exhibitedhigher hypoglycaemic action than that of the isoaspartate analogue asfound during iv injection with overnight-fasted Zucker fatty (fa/fa)rats (FIG. 8).

Persons skilled in this art will appreciate that variations andmodifications may be made to the invention as broadly described herein,other than those specifically described without departing from thespirit and scope of the invention. It is to be understood that thisinvention extends to include all such variations and modifications.

References:

-   1. Donahue R. P., Abbott R. D. Reed D. M. , Katsuhiko Y., (1987)    Diabetes 36, 689-692.-   2. Fuller, H. H., Shipley, M. J., Rose, G., Jarrett, R. J., Keen,    H., (1980) Lancet 1, 1373-1376.-   3. Jarrett, R. J., McCartney, P. and Keen, H., (1982) Diabetologia    22, 79-84.-   4. Report of the Expert Committee on the Diagnosis and    Classification of Diabetes Mellitus.

Diabetes Care 1997 July; 20(7): 1183-97.

-   5. Amos A F, McCarty D J, and Zimmet, (1994) Diabetic Medicine    Volume 14 (Supplement 5).-   6. Isaksson O. G. P, Schwartz J., Kostyo, J. L., and Reagan, C.    R., (1978) Endocrinology 105:452-458.-   7. Isaksson, O. G. P., Eden, S, and Jansson, J., (1985), Ann. Rev.    Physiol. 47:483-499-   8. Ng, F. M., (1993), Expr. Clin. Endocrinol (Life Sci. Adv.)    12:129-139-   9. Lim, N., Ng, F. M., Wu, Z. M., Ede, N. and Heam, M. T. W., (1992)    Endocrinology, 131, 835-840.-   10. Zimmet, P. Z., PhD Thesis, Monash University (1973)-   11. Ng, F. M., Zimmet, P. Z., Swiler, G., Taft, P. and    Bomstein, J. (1974) Diabetes 23,950-56.-   12. Wellings, D. A., and Atherton, E., (1997) In Methods in    Enzymology, Vol. 289, pp 44-66-   13. Merrifield, R. B., (1963) J. Am. Chem. Soc., 85, 2149.-   14. Barany, G., Kreib-Cordinia, N., and Mullen, D. G., (1988), In    Encyclopedia of Polymer Science& Engineering, 2^(nd) Edition,    Vol. 12. pp 811-858-   15. Geiger, T. and Clarke, S., (1987) J. Biol. Chem. 262, 785-794.-   16. Stephenson, R. C., and Clarke, S., (1989), J. Biol Chem., 264    6164-6170-   17. Van Handel E, (1965) Anal Biochem., 11, 256-265.-   18. Thompson, P. E., Lim, N., Ede, N. J., NG, F. M., Rae, I. D., &    Heam, M., (1995) Drug Design & Discovery 13:55-72-   19. Murray, E. D., Jr. and Clarke, S. (1984) J. Biol Chem., *259,    10722-10732.-   20. Clarke, S. (1987) Int. J. Pept. Protein Res., 30, 808-821.-   21. Stephenson, R. C. and Clarke, S. (1989) J. Biol. Chem. 264,    6164-6170.-   22. Bernhard, S. A., Berger, A., Carter, J. H., Katchalski, E.,    Sela, M. and Shalitn,Y. (1962) J. Am. Chem. Soc., 84, 2421-2434.-   23. Folsch, G. (1966) Acta. Chem. Scand., 20, 459473.-   24. Piszkiewics, D., Landon, M., and Smith, E. L. (1970) Biochem.    Biophys. Res. Commun., 40, 1173-1178.-   25. Brennan, T. V. and Clarke, S. (1993) Protein Science, 2,    331-338.-   26. Fledelins, C., Johnson, A. H., Cloos, P. A. C., Bonde, M. and    Qvist, P. 91997) J. Biol Chem., 272, 9755-9763.

1. A peptide of the formula:(Xaa)_(n1)-Xaa₁-His-Thr-Asp-(Xaa)_(n2) (SEQ ID NO:2) wherein Xaa is anyamino acid; Xaa₁ is a hydrophobic amino acid; n₁ is 0-10; and n₂ is0-10.
 2. A peptide according to claim 1, wherein the hydrophobic aminoacid is selected from the group consisting of Gly, Ala, Leu, lie, Pheand Val.
 3. A peptide according to claim 1, of the formula:(Xaa)_(n1)-Gly-His-Thr-Asp-(Xaa)_(n2) or (SEQ ID NO: 3)(Xaa)_(n1)-Val-His-Thr-Asp-(Xaa)_(n2) (SEQ ID NO: 4)

wherein Xaa, n₁, and n₂ are as defined in claim
 1. 4. A peptideaccording to claim 1, selected from: Gly-His-Thr-Asp; and (SEO ID NO: 5)Val-His-Thr-Asp. (SEO ID NO: 6)


5. A peptide according to any of claims 1 to 4, wherein the amino acidsare in the L-form.
 6. The isolated tetrapeptide Gly-His-Thr-Asp (SEQ IDNO:5), in substantially purified form.
 7. A method of regulating in vivoblood glucose levels in a human or other mammal, which comprisesadministration to said human or other mammal of an effective amount of apeptide of the formula:(Xaa)_(n1)-Xaa₁-His-Thr-Asp-(Xaa)_(n2) (SEQ ID NO:2) wherein Xaa is anyamino acid; Xaa_(l) is a hydrophobic amino acid; n₁ is 0-10; and n₂ is0-10.
 8. A method according to claim 7, wherein the peptide is a peptideof the formula: (Xaa)_(n1)-Gly-His-Thr-Asp-(Xaa)_(n2) or (SEQ ID NO: 3)(Xaa)_(n1)-Val-His-Thr-Asp-(Xaa)_(n2) (SEQ ID NO: 4)

wherein Xaa, n₁ and n₂ are as defined in claim
 7. 9. A method accordingto claim 7, wherein the peptide is selected from: Gly-His-Thr-Asp; and(SEQ ID NO: 5) Val-His-Thr-Asp. (SEQ ID NO: 6)


10. A method according to claim 7, wherein the regulation of in vivoblood glucose levels is for treatment of diabetes, particularly Type 2diabetes, in a human.
 11. Use of a peptide of the formula:(Xaa)_(n1)-Xaa₁-His-Thr-Asp-(Xaa)_(n2) (SEQ ID NO:2) wherein Xaa is anyamino acid; Xaa_(l) is a hydrophobic amino acid; n₁ is 0-10;and n₂ is0-10, in the manufacture of a composition for regulating in vivo bloodglucose levels in a human or other mammal.
 12. Use according to claim11, wherein the peptide is a peptide of the formula:(Xaa)_(n1)-Gly-His-Thr-Asp-(Xaa)_(n2) or (SEQ ID NO: 3)(Xaa)_(n1)-Val-His-Thr-Asp-(Xaa)_(n2) (SEQ ID NO: 4)

wherein Xaa, n₁, and n₂ are as defined in claim
 11. 13. Use according toclaim 11, wherein the peptide is selected from: Gly-His-Thr-Asp; and(SEQ ID NO: 5) Val-His-Thr-Asp. (SEQ ID NO: 6)


14. Use according to claim 11, wherein the composition is for treatmentof diabetes, particularly Type 2 diabetes, in a human.
 15. Apharmaceutical composition for regulating in vivo blood glucose levelsin a human or other mammal, which comprises the peptide of the formula:(Xaa)_(n1)-Xaa₁-His-Thr-Asp-(Xaa)_(n2) (SEQ ID NO:2) wherein Xaa is anyamino acid; Xaa_(l) is a hydrophobic amino acid; n₁ is 0-10; and n₂ is0-10, together with one or more pharmaceutically acceptable carriersand/or diluents.
 16. A composition according to claim 15, wherein thepeptide is a peptide of the formula:(Xaa)_(n1)-Gly-His-Thr-Asp-(Xaa)_(n2) or (SEQ ID NO: 3)(Xaa)_(n1)-Val-His-Thr-Asp-(Xaa)_(n2) (SEQ ID NO: 4)

wherein Xaa, n,₁ and n₂ are as defined in claim
 15. 17. A compositionaccording to claim 15, wherein the peptide is selected from:Gly-His-Thr-Asp; and (SEQ ID NO: 5) Val-His-Thr-Asp. (SEQ ID NO: 6)